This application is the national phase of international application PCT/DK98/00193 filed May 15, 1998 which designated the U.S.
This invention relates to a hitherto unknown class of compounds which shows strong activity in inhibiting undesirable cell proliferation in e.g. skin cells and cancer cells, to pharmaceutical preparations containing these compounds, to dosage units of such preparations, and to their use in the treatment and prophylaxis of diseases characterized by abnormal cell differentiation and/or cell proliferation such as e.g. psoriasis and cancer.
The compounds of the present invention are represented by the general formula I 
or their tautomeric forms, the attachment to the pyridine ring being in the 3- or 4-position, in which formula R stands for one or more substituents which can be the same or different and are selected from the group consisting of: hydrogen, halogen, trifluoromethyl, carboxy, C1-C4 alkyl, alkoxy or alkoxy-carbonyl, nitro, amino or cyano and Q stands for C5-C14 divalent hydrocarbon radical which can be straight, branched, cyclic, saturated or unsaturated and X stands for carboxy, amino, tetrahydropyranyloxy, C1-C4 saturated or unsaturated alkoxycarbonylamino, alkoxycarbonyl or di(alkoxy)phosphinoyloxy.
If the present compounds contain one or more asymmetric carbon atoms, these compounds may form optical isomers or diastereoisomers. The present invention also comprises such isomers, and mixtures of same.
The present salts of the compounds of formula I may be formed with pharmaceutically acceptable inorganic or organic acids, such as hydrochloric, hydrobromic and hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, 4-toluenesulfonic acid, methanesulfonic acid, formic acid, acetic acid, propionic acid, citric acid, tartaric acid, succinic acid, benzoic acid and maleic acid.
The present salts of the compounds of formula I may also be formed with pharmaceutically acceptable, inorganic or organic bases. Salts formed with pharmaceutically acceptable, non-toxic bases may be alkali metal salts and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium salts, as well as salts with ammonia and suitable non-toxic amines, such as C1-C6-alkylamines, e.g. triethylamine, C1-C6 alkanolamines, e.g. diethanolamine or triethanolamine, procaine, cycloalkylamines, e.g. dicydohexylamine, benzylamines, e.g. N-methyl-benzylamine, Nthylbenzylamine, N-benzyl-2-phenylethylamine, N,Nxe2x80x2-di-benzylethylenediamine or dibenzylamine, and heterocyclic amines, e.g. morpholine, N-ethylpiperidine and the like.
Even if the present compounds are well absorbed after enteral administration, in some cases it can be advantageous to prepare suitable bioreversible derivatives of compounds of the invention, i.e. to prepare so-called prodrugs, preferably derivatives, the physicochemical properties of which leads to improved solubility at physiological pH and/or absorption and/or bioavailability of the compound in question.
Such derivatives are for instance pyridyl N-oxide derivatives of compounds of the invention, such compounds being prepared by oxidation of the pyridyl N by a suitable oxidising agent, e.g. with 3-chloroperbenzoic acid in an inert solvent, e.g. dichloromethane.
Other suitable methods to improve the physicochemical properties and/or solubility of the compounds concerned can be used as well.
N-Alkyl-Nxe2x80x2-cyano-Nxe2x80x3-pyridylguanidines, described in United Kingdom Patent No. 1,489,879, are potent potassium channel activators with a pronounced effect as pre-capillary vasodilators, reducing the total peripheral resistance in animals and in man, and are thus useful as anti-hypertensives. As stated in International Patent No. PCT/DK93/00291, filing date Sep. 13, 1993, Publication No. WO 94/06770 the introduction of aryloxy-containing radicals into the aliphatic groups from the above-cited U.K. Patent has led to structures showing more specific pharmacological effects on isolated tissues and cells and with no or a negligible effect on 86Rb-efflux from potassium channels, as compared with the established effect of compounds covered by the above-mentioned U.K. Patent.
The compounds of the present invention inhibit the proliferation of various tumour cell lines in cultures at lower concentrations than the known compounds, confer Table 1 below, thus making them potentially useful in antineoplastic chemotherapy.
The inhibition of turmour cell proliferation was studied using different types of human cancer cell lines. The cell lines under investigation were small cell lung carcinoma (NYH), non small cell lung carcinoma (NCI-H460), and breast cancer (MCF-7) using the following general procedure:
The cells were cultured in vitro for 24 hours in the presence of the compound under investigation. DNA synthesis was measured by incorporation of [3H]thymidine, and the median inhibitory concentrations (IC50) of the compounds were calculated.
The results show that the compounds of the present invention are able to inhibit the proliferation of tumour cells in vim at lower concentrations than the compounds A and B from PCT/DK93/00291.
The compounds of the invention are well tolerated and non-toxic and are exerting the described beneficial activities with no or minimal effect on the systemic blood pressure. In general, they may be administered by oral, intravenous, intraperitoneal, intranasal or transdermal routes.
The present invention also relates to methods for preparing the desired compounds of the general formula I. The compounds of the formula I may conveniently be prepared by standard procedures detailed in the art. The routes are outlined in the following reaction scheme. 
R, Q and X as defined above.
a) DCCD, NH2CN, Et3N, CH3CN, 9 days. (General procedure 1).
b) DMAP, Et3N, pyridine. (General procedure 2).
The present compounds are intended for use in pharmaceutical compositions which are useful in the treatment of the above mentioned diseases.
The amount required of a compound of formula (I) (hereinafter referred to as the active ingredient) for therapeutic effect will, of course, vary both with the particular compound, the route of administration and the mammal under treatment A suitable dose of a compound of formula (I) for systemic treatment is 0.1 to 400 mg per kilogram bodyweight, the most preferred dosage being 1.0 to 100 mg per kg of mammal bodyweight, for example 5 to 20 mg/kg; administered once or more times daily.
A daily dose (for adults) may amount to 1 mg to 10000 mg, preferably from 70-5000 mg, and in the veterinary practice correspondingly, in daily doses from 0.1 to 400 mg/kg bodyweight
While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. Conveniently, the active ingredient comprises from 0.1% to 99% by weight of the formulation. Conveniently, dosage units of a formulation contain between 0.5 mg and 1 g of the active ingredient. For topical administration, the active ingredient preferably comprises from 1% to 20% by weight of the formulation but the active ingredient may comprise as much as 50% w/w. Formulations suitable for nasal or buccal administration may comprise 0.1% to 20% w/w. for example about 2% w/w of active ingredient.
By the term xe2x80x9cdosage unitxe2x80x9d is meant a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active material as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.
The formulations, both for veterinary and for human medical use, of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefor and optionally other therapeutic ingredient(s). The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
The formulations include those in a form suitable for oral, rectal, parenteral (including subcutaneous, intramuscular, intravenous and intraperitoneal) administration.
The formulations may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, If necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be administered in the form of a bolus, electuary or paste.
Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and a carrier such as cocoa butter, or in the form of an enema.
Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient.
In addition to the aforementioned ingredients, the formulations of this invention may include one or more additional ingredients, such as diluents, buffers, flavouring agents, binders, surface active agents, thickners, lubricants, preservatives, e.g. methylhydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
The compositions may further contain other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, e.g. antineoplastic agents which may result in synergisticc effects on tumour cells.
The invention will now be further described in the following general procedures and examples:
The exemplified compounds I are listed in table 2.
All melting points are uncorrected. For 1H and 13C nuclear magnetic resonance (NMR) spectra (300 MHz) chemical shift values (xcex4) are quoted, unless otherwise specified, for deuterochloroform and hexadeuterodimethylsulfoxide solutions relative to internal chloroform (1H NMR xcex4 7.25, 13C NMR xcex4 76.81) or tetramethylsilane (xcex4 0.00). The value for a multiplet (m), either defined (doublet (d), triplet (t), quartet (q)) or not at the approximate mid point is given unless a range is quoted (s singlet, b broad). Chromatography was performed on silica gel. The following abbreviations and formulas are used: DCCD (N,Nxe2x80x2-dicyclohexylcarbodiimide), NH2CN (cyanamide), Et3N (triethylamine), CH3CN (acetonitnle), DMAP (dimethylaminopyridine), MeOH (methanol), CH2Cl2 (methylene-chloride), EtOAc (ethylacetate), NH3 (ammonia), CDCl3 (deuterochloroform) and DMSO-d6 (hexadeuterodimethylsulfoxide).
General Procedure 1: Conversion of Compounds of the General Formula II into Compounds of the General Formula I.
A compound of the general formula II (5 mmol) was suspended in acetonitrile (12 ml) and dicyclohexylcarbodiimide (10 mmol), cyanamide (10 mmol) and triethylamine (0.07 ml ) was added. The reaction mixture was stirred at room temperature for 9 days.
The reaction mixture was filtered and washed with acetonitrile. The crude product was purified by flash chromatography (Eluent: 0-7% MeOH in CH2Cl2) and crystallized from ether to give the product of general formula I as white crystals.
General Procedure 2: Coupling of Compounds of the General Formula III with Compounds of the General Formula IV resulting in Compounds of the General Formula I.
A compound of the general formula III (4 mmol), a compound of the general formula IV (5 mmol), triethylamine (0.12 ml) and 4-dimethylamino-pyridine (15 mg) were dissolved in pyridine (4 ml). The reaction mixture was stirred at 60xc2x0 C. for 11 hours and then cooled to room temperature.
The product was precipitated with ether. Filtration resulted in pure product of the general formula I as white crystals.